Measurement of benzoylecgonine and cocaine in urine, separation of various cocaine metabolites using reversed-phase high-performance liquid chromatography

The application of reversed-phase high-performance liquid chromatography to the measurement of benzoylecgonine and cocaine in urine is described. Following a simple extraction and clean-up procedure, chromatography is performed using a column containing an octadecylsilica coated packing, elution with 17% acetonitrile in pH 2.7 phosphate buffer and ultraviolet detection at 200 or 235 nm. The detection limit is ca.0.1 microgram of drug per ml urine, and using the ethyl ester of benzoylecgonine as an internal standard, benzoylecgonine and cocaine are quantified with coefficients of variation of 7.0 and 2.8%, respectively. The procedure has been applied to urines from subjects receiving intranasal cocaine, and compared to the enzyme multiplied immunoassay technique. The chromatography procedure also permits the separation of norcocaine and benzoylnorecgonine.     

Determination of benzoylecgonine and cocaine in biologic fluids by automated gas chromatography

A method is described for extraction of the cocaine metabolite benzoylecgonine, conversion to the butyl ester derivative and gas chromatographic analysis using packed or capillary columns. Using a capillary column, cocaine and benzoylecgonine may be determined simultaneously. The extraction scheme has been designed to facilitate processing of large numbers of samples generated in pharmacokinetic studies. Structural analogues, m-toluylecgonine and m-toluylecgonine methyl ester, are used as internal standards. Concentrations as low as 10 ng/ml in 1-ml samples of plasma or urine are readily determined. Between-run coefficients of variation were 1.01% for cocaine and 4.18% for benzoylecgonine for concentrations of 75 and 350 ng/ml, respectively.     

Determination of cocaine and its metabolites using cation-selective exhaustive injection and sweeping-MEKC

We have employed a high-sensitivity on-line preconcentration method, cation-selective exhaustive injection (CSEI) and sweeping MEKC, for the analysis of cocaine, benzoylecgonine, norcocaine, and cocaethylene. We monitored the effects of several of the CSEI-sweeping-MEKC parameters - including the pH, the concentrations of SDS and organic modifier, the injection length of the high-conductivity buffer, and the injection time of the sample - to optimize the separation process. The optimal BGE was 100 mM phosphoric acid (pH 1.8) containing 75 mM SDS with 10% 2-propanol and 10% tetrahydrofuran as the organic modifier. In addition, electrokinetic injection of the sample at 15 kV for 900 s provided both high separation efficiency and enhanced sweeping sensitivity. The sensitivity enhancements for cocaine, norcocaine, and cocaethylene ranged from 2.06 x 10(4) to 3.96 x 10(4); for benzoylecgonine it was 1.75 x 10(3); the coefficients of determination exceeded 0.9958. The LODs, based on an S/N ratio of 3:1, of sweeping-MEKC ranged from 33.5 to 52.8 ng/mL; in contrast, when using CSEI-sweeping-MEKC the sensitivity increased to range from 29.7 to 236 pg/mL. Under the optimal conditions, we analyzed cocaine in a human urine sample prepared using off-line SPE to minimize the influence of the matrix. The recovery of the SPE efficiency was satisfactory (ca. 74.9-87.6%). Our experimental results suggest that, under the optimal conditions, the CSEI-sweeping-MEKC method can be used to determine cocaine and its metabolites with high sensitivity in human urine.     

Concentration profiles of cocaine, pyrolytic methyl ecgonidine and thirteen metabolites in human blood and urine: determination by gas chromatography-mass spectrometry

When cocaine is smoked, a pyrolytic product, methyl ecgonidine (anhydroecgonine methyl ester), is also consumed with the cocaine. The amount of methyl ecgonidine formed depends on the pyrolytic conditions and composition of the illicit cocaine. This procedure describes detection of cocaine and 10 metabolites--cocaethylene, nor-cocaine, nor-cocaethylene, methyl ecgonine, ethyl ecgonine, benzoylecgonine, nor-benzoylecgonine, m-hydroxybenzoylecgonine, p-hydroxybenzoylecgonine and ecgonine--in blood and urine. In addition, the detection of pyrolytic methyl ecgonidine and three metabolites--ecgonidine (anhydroecgonine), ethyl ecgonidine (anhydroecgonine ethyl ester) and nor-ecgonidine (nor-anhydroecgonine)--are included. The newly described metabolites, ethyl ecgonidine and nor-ecgonidine, were synthesized and characterized by gas chromatography-mass spectrometry (GC-MS). All 15 compounds were extracted from 3 mL of blood or urine by solid-phase extraction and identified by a GC-MS method. The overall recoveries were 49% for methyl ecgonine, 35% for ethyl ecgonine, 29% for ecgonine and more than 83% for all other drugs. The limits of detection were between 0.5 and 4.0 ng/mL except for ecgonine, which was 16 ng/mL. Linearity for each analyte was established and in all cases correlation coefficients were 0.9985-1.0000. The procedure was applied to examine the concentration profiles of analytes of interest in post-mortem (PM) blood and urine, and in urine collected from living individuals (LV). These specimens previously were shown to be positive for the cocaine metabolite, benzoylecgonine. Ecgonidine, the major metabolite of methyl ecgonidine, was present in 77% of PM and 88% of the LV specimens, indicating smoking as the major route of cocaine administration. The new pyrolytic metabolites, ethyl ecgonidine and nor-ecgonidine, were present in smaller amounts. The urine concentrations of nor-ecgonidine were 0-163 ng/mL in LV and 0-75 ng/mL in PM specimens. Ethyl ecgonidine was found only in PM urine at concentrations 0-39 ng/mL. Ethanol-related cocaine metabolites, ethyl ecgonine or cocaethylene, were present in 69% of PM and 53% of cocaine-positive LV specimens, implying alcohol consumption with cocaine use. The four major metabolites of cocaine--benzoylecgonine, ecgonine, nor-benzoylecgonine and methyl ecgonine--constituted approximately 88 and 97% of all metabolites in PM and LV specimens, respectively. The concentrations of nor-cocaine and nor-cocaethylene were consistently the lowest of all cocaine metabolites. At benzoylecgonine concentrations below 100 ng/mL, ecgonine was present at the highest concentrations. In 20 urine specimens, benzoylecgonine and ecgonine median concentrations (range) were 54 (0-47) and 418 ng/mL (95-684), respectively. Therefore, detection of ecgonine is advantageous when benzoylecgonine concentrations are below 100 ng/mL.     

Simultaneous analysis of cocaine and its metabolites in urine by capillary electrophoresis-electrospray mass spectrometry using a pressurized liquid junction nanoflow interface

A new method for the simultaneous separation of cocaine and four metabolites in urine by CE-ESI-MS via a pressurized nanoliquid junction interface was developed. The resolution of cocaine, cocaethylene, benzoylecgonine, norcocaine, and ecgonine methyl ester was achieved in a polyvinyl-alcohol-coated capillary with 75 μm id × 50 cm total length, using a 15 mM ammonium formate electrolyte solution (pH 9.5) in less than 15 min. In addition, to enhance sensitivity, a field-amplified sample injection (FASI) was evaluated in terms of injection time and sample solvent composition. The limits of detection achieved with the FASI method ranged from 1.5 to 10 ng/mL for all the compounds. The detection of the studied compounds was performed using an ion-trap mass spectrometer in a positive ionization mode. A mixture of methanol:water (80:20 v/v) containing 0.1% v/v of formic acid was employed as spray liquid and delivered at ~200 nL/min. Under optimal CE-MS conditions, linearity was assessed in the concentration range of interest for all analytes with correlation coefficients r2 ≥ 0.9913. Intra- and inter-day precision provided a relative standard deviation lower than 1.54% for migration times and lower than 12.15% for peak areas. Finally, urine samples, spiked with the standard mixture, were extracted using a solid-phase extraction procedure and injected under FASI conditions, providing recoveries from 80% to 94% for all analytes.     

Surface-activated chemical ionization ion trap mass spectrometry for the analysis of cocaine and benzoylecgonine in hair after extraction and sample dilution

Surface-activated chemical ionization (SACI) was employed for the analysis of cocaine and its metabolite, benzoylecgonine, extracted from hair. Following decontamination and acid hydrolysis procedures on the hair sample, the sample solution was diluted (1:10) and directly analyzed by liquid chromatography/surface-activated chemical ionization multiple collisional stage single reaction monitoring mass spectrometry (LC/SACI-MS(3)-SRM) without solid-phase extraction (SPE) pre-purification and concentration procedures. To increase the selectivity of the method, MS(3) was chosen instead of the less selective MS/MS. This data was compared with that achieved using gas chromatography/mass spectrometry (GC/MS), the reference method used by the Italian Government Institute of Health protocol. The limits of detection (LODs) were 0.003 ng/(mg hair) for cocaine and 0.02 ng/(mg hair) for benzoylecgonine and the limits of quantitation (LOQs) were 0.01 ng/(mg hair) for cocaine and 0.04 ng/(mg hair) for benzoylecgonine. The squared correlation coefficient (R(2)) of the calibration curve was 0.9887-0.9980 for cocaine and 0.9987-0.9997 for benzoylecgonine. The percent accuracy error was 2-5% for both cocaine and benzoylecgonine using the LC/SACI-MS(3)-SRM approach, whereas it was higher for benzoylecgonine (20-25%) using the LC/SACI-MS/MS-SRM approach compared with the GC/MS data due to hair matrix contamination. In both cases, high precision was achieved (1-3% precision error), which confirmed the stability of the developed methods.     

Determination of cocaine and norcocaine in plasma and cell cultures using high-performance liquid chromatography

A new simple high-performance liquid chromatographic (HPLC) method was developed for the determination of cocaine and norcocaine. Cocaine and norcocaine in biological samples were buffered to pH 9.0, extracted with diethyl ether and reextracted in a 0.1% aqueous solution of tetramethylammonium hydrogen sulfate (TMAHS) with a theoretical yield of extraction of 100%. The HPLC elution of cocaine and norcocaine was performed using a Spherisorb RP-18, 100 mm x 4.6 mm I.D., 5 microns particle size column with a mobile phase containing acetonitrile-0.1% TMAHS aqueous solution (60:40). The compounds were entirely separated, and a reliable limit of quantitation was set at 20 ng/ml when extracted from 0.5 ml of plasma. No interference with 26 other drugs was found. Cocaine and norcocaine stability studies showed that their half-lives in human plasma incubated at 37 degrees C were 50.8 and 43.2 min, respectively. In contrast, plasma from dogs or rats exhibited only weak or no enzymatic esterase activity towards cocaine and norcocaine resulting in less rapid degradation. Hydrolysis could be efficiently inhibited with sodium fluoride and prevented by storage of the sample at -20 degrees C. The highly sensitive assay also allowed the assessment of the oxidative metabolism pathway of cocaine to norcocaine in primary rat hepatocyte cultures.     

Development and validation of an analytical method for the simultaneous determination of cocaine and its main metabolite, benzoylecgonine, in human hair by gas chromatography/mass spectrometry

A new, simple and rapid procedure has been developed and validated for the determination of cocaine and its main metabolite, benzoylecgonine, in human hair samples. After extraction from within the hair matrix by a mixture of methanol/hydrochloric acid (2:1) at 65 degrees C for 3 h, and sample cleanup by mixed-mode solid-phase extraction (SPE), the extracts were analyzed by gas chromatography/mass spectrometry (GC/MS), after derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide with 5% chlorotrimethylsilane. Using a sample size of only 20 mg of hair, limits of detection (LODs) and quantitation (LOQs) were, respectively, 20 and 50 pg/mg for cocaine, and 15 and 50 pg/mg for benzoylecgonine, achieving the cut-off values proposed by the Society of Hair Testing for the analysis of these compounds in hair. The method was found to be linear (weighing factor of 1/x) between the LOQ and 20 ng/mg for both compounds, with correlation coefficients ranging from 0.9974 to 0.9996 for cocaine; and from 0.9981 to 0.9994 for benzoylecgonine. Intra- and interday precision and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. The sample cleanup step presented a mean absolute recovery greater than 90% for both compounds. The developed method may be useful in forensic toxicology laboratories for the analysis of cocaine and benzoylecgonine in hair samples, taking into account its speed (only 3 h are required for the extraction of the analytes from within the matrix, whereas 5 h or even overnight extractions have been reported) and the low limits achieved (using a single quadrupole mass spectrometer, which is available in most laboratories).     

Profiling of cocaine by micellar electrokinetic chromatography

The potential of micellar electrokinetic chromatography (MEKC) for the profiling of cocaine samples is described. An MEKC system containing sodium dodecyl sulfate (SDS) and methanol was optimized using a test mixture of cocaine, its common impurities (benzoylecgonine, norcocaine, tropacocaine, and trans-cinnamoylcocaine), and several degradation products. The effect of pH, percentage modifier, and concentration surfactant on the separation has been investigated. The optimal separation buffer for cocaine samples consisted of 75 mM SDS, 17.5% methanol, and 25 mM borate (pH 8.3) and was well suited to separate components of diverse polarity in one run. Various cocaine seizures have been analyzed with the MEKC system and their signatures were compared. The electrokinetic chromatograms obtained were characteristic, and differences and similarities among the samples could easily be observed. Several impurities were identified in the samples by means of migration times and comparison of recorded and library UV spectra. The composition of the samples was determined semiquantitatively using relative corrected peak areas.     

Determination of cocaethylene, cocaine and their metabolites in rat serum microsamples by high-performance liquid chromatography, and its application to pharmacokinetic studies in rodents.

A single-solvent extraction step high-performance liquid chromatographic method is described for quantitating cocaethylene in rat serum microsamples (50 microliters), a substance formed in vivo when cocaine and ethanol are present concurrently. The separation used a 2 mm I.D. reversed-phase Nova-Pak C18 column with a mobile phase of acetonitrile-phosphate buffer containing an ion-pairing reagent. With an ultraviolet detector operated at 230 nm, a linear response was observed from 0.05 to 2.0 micrograms/ml with a detection limit of 5 ng/ml for cocaethylene, cocaine and norcocaine. The method showed a longer half-life for cocaethylene than for cocaine in rat.     

Thin-layer chromatographic analysis of cocaine and benzoylecgonine in urine.

The sensitivity achieved by the described thin-layer chromatographic (TLC) method greatly exceeds that of previously published TLC methods for the determination of cocaine and its principal metabolite, benzoylecgonine, in urine. Sensitivity for cocaine and benzoylecgonine approaches 0.1 and 0.25 mug/ml, respectively, for a 5.0-ml specimen. A simple extraction with a mixed organic solvent provides the basic mechanism for isolating the drugs from biologic specimens. Cocaine and its metabolites are stable in sulfuric acid solutions but labile in aqueous media containing certain other inorganic and organic acids; therefore, an emphasis on the utilization of sulfuric acid solutions is employed throughout the procedure. An evaluation of sensitivities achieved for cocaine and benzoylecgonine by various detection reagents is presented. The technique is applicable to drug screening programs.     

Development and validation of two LC-MS/MS methods for the detection and quantification of amphetamines, designer amphetamines, benzoylecgonine, benzodiazepines, opiates, and opioids in urine using turbulent flow chromatography

In the context of driving ability diagnostics in Germany, administrative cutoffs for various drugs and pharmaceuticals in urine have been established. Two liquid chromatography–tandem mass spectrometry methods for simultaneous detection and quantification of amphetamines, designer amphetamines, benzoylecgonine, benzodiazepines, opiates, and opioids in urine were developed and validated. A 500-μL aliquot of urine was diluted and fortified with an internal standard solution. After enzymatic cleavage, online extraction was performed by an ion-exchange/reversed-phase turbulent flow column. Separation was achieved by using a reversed-phase column and gradient elution. For detection, a Thermo Fisher TSQ Quantum Ultra Accurate Mass tandem mass spectrometer with positive electrospray ionization was used, and the analytes were measured in multiple-reaction monitoring mode detecting two transitions per precursor ion. The total run time for both methods was about 15 min. Validation was performed according to the guidelines of the Society of Toxicological and Forensic Chemistry. The results of matrix effect determination were between 78 % and 116 %. The limits of detection and quantification for all drugs, except zopiclone, were less than10?ng/mL and less than 25 ng/mL, respectively. Calibration curves ranged from 25 to 200 ng/mL for amphetamines, designer amphetamines, and benzoylecgonine, from 25 to 250 ng/mL for benzodiazepines, from 12.5 to 100 ng/mL for morphine, codeine, and dihydrocodeine, and from 5 to 50 ng/mL for buprenorphine and norbuprenorphine. Intraday and interday precision values were lower than 15 %, and bias values within?±?15 % were achieved. Turbulent flow chromatography needs no laborious sample preparation, so the workup is less time-consuming compared with gas chromatography–mass spectrometry methods. The methods are suitable for quantification of multiple analytes at the cutoff concentrations required for driving ability diagnostics in Germany.     

Determination of the pyridinium metabolite derived from haloperidol in brain tissue, plasma and urine by high-performance liquid chromatography with fluorescence detection.

A sensitive and selective method for the determination of the pyridinium metabolite (HPP+) derived from the antipsychotic drug haloperidol (HP) in brain tissue, plasma and urine using high-performance liquid chromatography with fluorescence detection is described. The HPP+ present in biological samples was extracted using a Sep-Pak C18 cartridge. Recoveries of HPP+ ranged from 78 to 90%. Final separation and quantitative estimations of HPP+ were achieved on a C18 reversed-phase column employing a mobile phase of acetonitrile-30 mM ammonium acetate (40:60, v/v) containing 10 mM triethylamine and adjusted to pH 3 with trifluoroacetic acid. The fluorescence detection utilized an excitation wavelength of 304 nm and an emission wavelength of 374 nm. Standard curves were linear in the range of 2.5-100 ng/ml for brain tissue homogenate and plasma samples and 10-500 ng/ml for urine samples. The detection limit of HPP+ was about 1 ng/ml in all biological samples. The concentrations of HPP+ in brain tissue, plasma and urine from HP-treated rats were determined using this method.     

Multi-residue screening of bovine urine on xenobiotic oestrogens with an oestrogen radioreceptor assay

An improved radioreceptor assay (RRA) is used for the screening of urine samples from cattle for the presence of exogenous oestrogenic anabolic compounds, e.g., stilbenes and zeranol. The method includes extraction of the hormones from urine samples with simultaneous purification using reversed-phase C18 cartridges. High-performance liquid chromatography is used to isolate the anabolics from the naturally occurring oestrogens. Fractions containing the stilbenes and zeranol are collected and subsequently analysed using the RRA with the oestrogen receptor, isolated from immature calf uteri, as a binder and tritiated 17 beta-oestradiol as a tracer. Urine samples from untreated calves and cows and samples from calves treated with zeranol-trenbolon acetate, dienoestrol or hexoestrol or samples containing diethylstilboestrol were analysed with this RRA method. Sensitivity, specificity and predictive values were calculated at different classification levels (0.4, 0.5, 0.6 and 1.0 ng/ml 'apparent' oestradiol in urine). An optimum sensitivity (89%) with a maximum specificity (95%) was reached at a classification level of 0.6 ng/ml. At this level the detection limits in urine samples are 0.5 ng/ml for hexoestrol, 0.6 ng/ml for diethylstilboestrol, 0.9 ng/ml for dienoestrol and 5.0 ng/ml for zeranol.     

Simultaneous determination of cocaine and its metabolites with caffeine in rat serum microsamples by high-performance liquid chromatography

A single, isocratic high-performance liquid chromatographic method is described for the determination of cocaine and three of its metabolites along with caffeine in serum microsamples (50 microliters). The small sample size permits the tracking of pharmacokinetic data over time in individual, small animals. The method also was used to demonstrate that cocaine, benzoylecgonine and norcocaine in rat serum samples were stable for at least a month without the presence of sodium fluoride.     

Screening of Benzoylecgonine in Urine by Cyclobond Solid Phase Extraction and High Performance TLC

Abstract

A method is described for screening of the cocaine metabolite benzoylecgonine in urine at 0.2 μ/ml using a Cyclobond cyclodex^?trin solid phase extraction cartridge and high performance thin layer chromatography. Results are compared with those obtained using liquid-liquid and diatomaceous earth column extraction and C-18 solid phase extraction.     

The Determination of a New Trifluorinated Quinolone,Fleroxacin, Its N-Demethyl,and N-Oxide Metabolites in Plasma and Urine by High Performance Liquid Chromatography with Fluorescence Detection

Abstract

A liquid chromatographic method is described for the determination of the new fluoroquinolone Ro 23–6240 and its N-demethyl and N-oxide metabolites in plasma and urine. The three substances were extracted from aqueous solution with dichloromethane/isopropanol containing sodium dodecyl sulphate. After evaporation and reconstitution, samples were analysed on a reversed-phase column using ion pair chromatography and fluorescence detection. The limit of quantification was 10–20 ng/ml (RSD 4%) using a 0.5 ml plasma sample, and the inter assay precision was 3–10% over the concentration range 50 ng/ml to 20 μg/ml. Recovery from plasma was 81% (RSD 10%) over the range 10 ng/ml to 5 μg/ml. The method has been applied successfully to the analysis of several thousand samples from human pharmacokinetic studies. Care has to be taken to avoid exposure of samples to direct sunlight, and the use of opaque vessels for sample storage and handling is recommended.     

Analysis of anabolic steroids in hair by GC/MS/MS

A simple and sensitive gas chromatography/tandem mass spectrometry (GC/MS/MS) method is described for the detection of anabolic steroids, usually found in keratin matrix at very low concentrations. Hair samples from seven athletes who spontaneously reported their abuse of anabolic steroids, and in a single case cocaine, were analyzed for methyltestosterone, nandrolone, boldenone, fluoxymesterolone, cocaine and its metabolite benzoylecgonine. Anabolic steroids were determinate by digestion of hair samples in 1 m NaOH for 15 min at 95 degrees C. After cooling, samples were purificated by solid-phase and liquid-liquid extraction, then anabolic steroids were converted to their trimethylsilyl derivative and finally analyzed by GC/MS/MS. For detection of cocaine and benzoylecgonine, hair samples were extracted with methanol in an ultrasonic bath for 2 h at 56 degrees C then overnight in a thermostatic bath at the same temperature. After the incubation, methanol was evaporated to dryness, and benzoylecgonine was converted to its trimethylsilyl derivative prior of GC/MS/MS analysis. Results obtained are in agreement with the athletes' reports, confirming that hair is a valid biological matrix to establish long-term intake of drugs.     

Comprehensive automation of the solid phase extraction gas chromatographic mass spectrometric analysis (SPE-GC/MS) of opioids,cocaine, and metabolites from serum and other matrices

The analysis of opioids, cocaine, and metabolites from blood serum is a routine task in forensic laboratories. Commonly, the employed methods include many manual or partly automated steps like protein precipitation, dilution, solid phase extraction, evaporation, and derivatization preceding a gas chromatography (GC)/mass spectrometry (MS) or liquid chromatography (LC)/MS analysis. In this study, a comprehensively automated method was developed from a validated, partly automated routine method. This was possible by replicating method parameters on the automated system. Only marginal optimization of parameters was necessary. The automation relying on an x-y-z robot after manual protein precipitation includes the solid phase extraction, evaporation of the eluate, derivatization (silylation with N-methyl-N-trimethylsilyltrifluoroacetamide, MSTFA), and injection into a GC/MS. A quantitative analysis of almost 170 authentic serum samples and more than 50 authentic samples of other matrices like urine, different tissues, and heart blood on cocaine, benzoylecgonine, methadone, morphine, codeine, 6-monoacetylmorphine, dihydrocodeine, and 7-aminoflunitrazepam was conducted with both methods proving that the analytical results are equivalent even near the limits of quantification (low ng/ml range). To our best knowledge, this application is the first one reported in the literature employing this sample preparation system.     

Extraction, quantitative gas chromatographic determination and gas chromatographic/mass spectrometric detection of ecgonine for identification of cocaine and its metabolites in urine

Summary Quantitative extraction studies from urine were carried out by addition of cocaine, benzoylecgonine, ecgonine methyl ester and ecgonine to urine samples. After hydrolysis to ecgonine the compounds were analyzed together. Ecgonine was isolated by a cation-exchange resin and purified by an anion-exchange resin. The quantitative determination was performed by GC after silylation with MSTFA. The recovery was 77% at a concentration of 150 g ecgonine/ml urine. A qualitative determination of ecgonine by GC/MS was possible up to the detection limit of 20 ng/ ml. The method can be applied for the detection of cocaine abuse.

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Extraktion, quantitative gas-chromatographische Bestimmung und gas-chromatographischer massenspektrometrischer Nachweis von Ecgonin zur Identifizierung von Cocain und seinen Metaboliten in Urin

Zusammenfassung Es wurden quantitative Untersuchungen zur Extraktion von Urin durchgeführt, dem Cocain, Benzoylecgonin, Ecgoninmethylester und Ecgonin zugesetzt wurden. Die Summe dieser Verbindungen kann über eine vorgeschaltete Hydrolyse zu Ecgonin indirekt bestimmt werden. Nach Hydrolyse wird Ecgonin an einem Kationenaustauscher isoliert und der Extrakt an einem Anionenaustauscher gereinigt. Die quantitative Bestimmung erfolgt gaschromatographisch nach Silylierung mit MSTFA. Dabei beträgt die Wiederfindungsrate 77% bei Konzentrationen von 150 g Ecgonin/ml Urin. Der qualitative Nachweis von Ecgonin durch GC/MS ist bis zu einer Nachweisgrenze von 20 ng/ml möglich. Damit eignet sich die Methode zum Nachweis einer Cocain-Einnahme im Urin.

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From the dissertation by U. Zerell, University of Bonn, 1986 (in preparation)